Neurospora Importin Î± Is Required for Normal ... - PLOS

RESEARCH ARTICLE

Neurospora Importin α Is Required for Normal Heterochromatic Formation and DNA Methylation Andrew D. Klocko, Michael R. Rountree¤a, Paula L. Grisafi, Shan M. Hays¤b, Keyur K. Adhvaryu¤c, Eric U. Selker* Institute of Molecular Biology, University of Oregon, Eugene, Oregon, United States of America ¤a Current address: Nzumbe Inc., Portland, Oregon, United States of America ¤b Current address: Natural and Environmental Sciences Department, Western State Colorado University, Gunnison, Colorado, United States of America ¤c Current address: Department of Biology, York University, Toronto, Ontario, Canada * [email protected]

OPEN ACCESS Citation: Klocko AD, Rountree MR, Grisafi PL, Hays SM, Adhvaryu KK, Selker EU (2015) Neurospora Importin α Is Required for Normal Heterochromatic Formation and DNA Methylation. PLoS Genet 11(3): e1005083. doi:10.1371/journal.pgen.1005083 Editor: Eduardo A. Espeso, Centro de Investigaciones Biológicas, Spain Received: September 15, 2014 Accepted: February 19, 2015 Published: March 20, 2015 Copyright: © 2015 Klocko et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Data files of H3K9me3 ChIP-seq and Bisulfite-seq have been deposited to GEO (http://www.ncbi.nlm.nih.gov/geo/) under the accession numbers (GSE61173; ChIP-seq) and (GSE61174; BS-seq). The accession number GSE61175 reports both data sets. All other data are presented within the paper and its Supporting Information files. Funding: This work was funded by grants from the US National Institutes of Health to EUS (GM035690) and ADK (GM097821). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Abstract Heterochromatin and associated gene silencing processes play roles in development, genome defense, and chromosome function. In many species, constitutive heterochromatin is decorated with histone H3 tri-methylated at lysine 9 (H3K9me3) and cytosine methylation. In Neurospora crassa, a five-protein complex, DCDC, catalyzes H3K9 methylation, which then directs DNA methylation. Here, we identify and characterize a gene important for DCDC function, dim-3 (defective in methylation-3), which encodes the nuclear import chaperone NUP-6 (Importin α). The critical mutation in dim-3 results in a substitution in an ARM repeat of NUP-6 and causes a substantial loss of H3K9me3 and DNA methylation. Surprisingly, nuclear transport of all known proteins involved in histone and DNA methylation, as well as a canonical transport substrate, appear normal in dim-3 strains. Interactions between DCDC members also appear normal, but the nup-6dim-3 allele causes the DCDC members DIM-5 and DIM-7 to mislocalize from heterochromatin and NUP-6dim-3 itself is mislocalized from the nuclear envelope, at least in conidia. GCN-5, a member of the SAGA histone acetyltransferase complex, also shows altered localization in dim-3, raising the possibility that NUP-6 is necessary to localize multiple chromatin complexes following nucleocytoplasmic transport.

Author Summary The epigenetic information contained in chromatin is essential for development of higher organisms, and if misregulated, can lead to the unregulated growth associated with human cancers. Chromatin is typically classified into two basic types: gene-rich 'euchromatin', and gene-poor heterochromatin, which is also rich in repeated DNA and 'repressive chromatin marks'. As in humans and eukaryotes generally, heterochromatin in Neurospora crassa is decorated with DNA methylation and histone H3 lysine 9 (H3K9) methylation,

PLOS Genetics | DOI:10.1371/journal.pgen.1005083

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A Role for Importin α in Heterochromatin Formation

Competing Interests: The authors have declared that no competing interests exist.

but unlike the case in mammals, loss of these epigenetic marks does not compromise viability. In Neurospora, the DCDC, a five-member Cul4-based protein complex, trimethylates H3K9. Little information is available on the regulation of DCDC or similar complexes in other organisms. Using forward genetics, we identified a novel role for Importin α (NUP-6) for the function of DCDC. Although NUP-6 typically functions in nucleocytoplasmic transport, the dim-3 strain, which contains an altered nup-6 gene that reduces DNA methylation and H3K9me3, shows normal nuclear transport of the heterochromatin machinery and a canonical transport substrate. Two DCDC members are mislocalized from heterochromatin in the dim-3 mutant, signifying that NUP-6 may be important for targeting key proteins to incipient heterochromatic DNA. The euchromatic complex SAGA has increased euchromatin localization in dim-3, suggesting that NUP-6 may localize multiple chromatin complexes to sub-nuclear genomic targets.

Introduction The densely staining regions of eukaryotic chromosomes, referred to as heterochromatin, typically contain repetitive, A:T rich DNA, and are characterized by low gene density, reduced genetic recombination, di- or tri-methylation of lysine 9 on histone H3 (H3K9me2 or H3K9me3), and DNA methylation [1–4]. Heterochromatin is critical for centromere and telomere function, and is largely responsible for the silencing of transposable elements [1,4]. Unlike the situation in animals and plants, which require DNA methylation for normal development [5,6], the fungus Neurospora crassa does not require DNA methylation for viability [4,7]. Neurospora has characteristics of heterochromatin found in higher eukaryotes and is convenient for genetic and biochemical studies. These traits have led to the identification of genes involved in establishing, maintaining, and regulating DNA methylation and other features of heterochromatin [8] A single DNA methyltransferase (DNMTase), DIM-2, is responsible for all DNA methylation in vegetative tissue of Neurospora [9]. DIM-2 directly interacts with heterochromatin protein-1 (HP1) [10], which binds to H3K9me3 [11,12]. The histone methyltransferase (HMTase) DIM-5 [13,14] is responsible for trimethylation of H3K9. In vivo, DIM-5 activity depends on all members of the five protein complex, DCDC (DIM-5/-7/-9, CUL4, DDB1 Complex) but DIM-7 alone appears sufficient to target DIM-5 to incipient heterochromatin regions [8,15]. DCDC resembles Cullin-4 E3 ubiquitin ligase complexes, with the WD-40 protein DIM-9 being the putative DCAF (DDB1/CUL4 associated factor), which is normally expected to recognize substrates destined for ubiquitination. However, results of recent studies indicate that DCDC does not function as a canonical ubiquitin ligase [16]. Thus, important questions regarding how DCDC and other members of the heterochromatin/DNA methylation machinery function and are controlled remain unanswered. To improve our understanding of the control of DNA methylation and heterochromatin formation, we characterized the Neurospora dim-3 strain, which shows a substantial loss of DNA methylation [7]. Genetic mapping, whole genome sequencing, and complementation tests identified the causative mutation in the nup-6 gene, resulting in a critical change in the eighth ARM repeat of NUP-6 (Importin α). This protein (also known as Srp1p in yeast and karyopherin α in humans) is the canonical nucleocytoplasmic transport adaptor. Importin α binds “cargo” proteins to be transported into the nucleus, complexes with Importin β, and then is shuttled through the nuclear pore complex to the nucleoplasm [17–19]. We found that dim3 strains have a drastic reduction in global H3K9me3, indicating that NUP-6 is required for


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proper DCDC function. Nuclear transport and interactions of critical DCDC components appear normal in dim-3 strains but at least two DCDC components (DIM-5 and DIM-7) are mislocalized from heterochromatin. Curiously, the SAGA histone acetyltransferase, but not DCDC components, showed increased localization in euchromatin of a dim-3 strain. Altogether, our results reveal a nuclear transport-independent role of NUP-6 in localizing chromatin complexes to sub-nuclear targets.

Results Loss of DNA methylation in dim-3 strains is caused by a mutation in the nup-6 gene The dim-3 gene was genetically identified in a brute-force screen for methylation defects following N-methyl-N’-nitro-N-nitrosoguanidine mutagenesis [7]. Southern blots probed for the representative interspersed heterochromatic regions 8:G3, 8:A6, and 2:B3 [20] in a histidine auxotrophic dim-3 strain illustrate the substantial DNA methylation reduction caused by the allele (Fig. 1A). Genome-wide bisulfite sequencing (BS-Seq; Rountree and Selker, in preparation) demonstrated that the residual DNA methylation in a dim-3 mutant is distributed normally, or largely normally, to heterochromatic regions (Figs. 1B and S1). Dim-3 was mapped to the right arm of Linkage Group V, and high-throughput sequencing identified two point mutations in the open reading frame of gene NCU01249, E396K and R469H; both were confirmed by Sanger sequencing. NCU01249 encodes NUP-6 (NIH GenBank accession EAA31416.1), which is predicted to form a structure similar to yeast Importin α [21], a protein that is highly conserved in eukaryotes [19]. Neurospora NUP-6 includes an N-terminal *80 amino acid Importin β-binding domain (IBB), followed by ten *40 amino acid ARM repeats (Fig. 1C), each of which should fold into a triple-alpha helical bundle to form a binding pocket for the nuclear localization signals (NLS) of cargo proteins (S2A Fig.; [22]). ARM repeats 1–9 are thought to recognize the NLS [22] while cargo release factors interact with the less-conserved 10th ARM repeat [23]. The E396K and R469H changes in nup-6dim3 are in the putative 8th and 10th ARM repeats, respectively (Figs. 1C and S2A), and should not destabilize the NUP-6dim-3 protein nor impact its nuclear shuttling (S2B-S2C Fig.). Interestingly, dim-3 strains have a minor growth defect (S2D Fig.) and are homozygous sterile, indicating that one or both of the amino acid substitutions compromise an important cellular process. To confirm that nup-6dim-3 causes the observed DNA methylation loss in dim-3 strains, we introduced a wild type (WT) nup-6 gene (nup-6+) at an ectopic locus (his-3) of a dim-3 strain. Ectopic nup-6+ restored global DNA methylation to WT levels at all the representative heterochromatic loci tested (Fig. 1A), suggesting that nup-6dim-3 was indeed responsible for the Dim phenotype. To determine if one or both of the mutations cause the phenotype, we replaced the endogenous nup-6+ allele with engineered mutant alleles. As expected, the strain with the reintroduced nup-6dim-3 allele showed reduced DNA methylation, although somewhat less so than the original dim-3 isolate (Figs. 1D and S3). Reintroduction of the R469H mutation did not affect DNA methylation, whereas the reintroduced E396K mutation caused a loss equivalent to that observed with the reintroduced nup-6dim-3 allele, implicating this change as the causative mutation (Figs. 1D and S3). As expected, the nup-6 gene appears essential, because strains with the gene deleted are only viable as heterokaryons, containing both nup-6+ and Δnup-6::hph nuclei [24]. Thus, there is no reason to expect that the E396K creates a null allele.


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Fig 1. The DNA methylation deficiency in dim-3 strains is caused by mutations in nup-6. [A]. Southern blots showing DNA methylation loss in a dim-3 strain and complementation by nup-6+ at the his-3 locus. The ectopic his-3+::nup-6+ strain was grown in the presence of histidine for comparison with the auxotrophic dim-3 strain. Genomic DNA from dim-3+ (Wild type [“WT”]), dim-5, dim-3; his-3, and dim-3; his-3+::nup-6+ strains was digested with the 5mCinsensitive restriction endonuclease DpnII (D) or its 5mC-sensitive isoschizomer BfuCI (B), fractionated on an agarose gel, transferred to a nylon membrane, and probed for heterochromatic regions (8:A6, 8:G3, and 2:B3; [20]) or an unmethylated region (am). The ethidium bromide (EtBr)-stained gel, with the positions of size markers (Kb) indicated, is shown because it provides an indication of global differences in DNA methylation. A reported restriction site polymorphism in the 2:B3 region is evident for the dim-5 strain [11]. [B] Bisulfite sequencing (BS-seq) of methylated cytosines from dim-3+ (Wild type, “WT”, black track) and dim-3 strains (grown in minimal medium = red track, grown in medium containing histidine = blue track) displayed by the Integrative Genomics Viewer, with y-axis denoting the number of normalized mapped reads (reads*106/total number of mapped reads; [58]). Linkage Group II (LGII) is shown, and two heterochromatic peaks are displayed at higher magnification below. Genes are displayed on the x-axis below the 5mC peaks as vertical lines, while distance (in Megabases) from the left end of LG II is displayed above the graph. Due to the reduced cytosine methylation in the dim-3 samples, the signal-to-noise ratio is lower, which results in disproportionally high background peaks. [C] Schematic of NUP-6 structure, with ARM repeats 1–10, the Nterminal Importin β binding domain (IBB), and the mutations found in the dim-3 allele indicated (penetrant mutation, E396K, is shown in black). [D] Southern blot assay of indicated mutations reintroduced into a dim-3+ (WT) strain, along with dim-5, dim-3, and WT controls. doi:10.1371/journal.pgen.1005083.g001

Histidine exacerbates the dim-3-mediated DNA methylation loss While comparing DNA methylation levels in various dim-3 strains, we noticed that the histidine-requiring dim-3 strains had an exacerbated reduction in DNA methylation, leading us to


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systematically test the possible effect of histidine on DNA methylation in dim-3 and dim-3+ strains. We found that histidine supplementation decreased DNA methylation at all heterochromatic regions tested in a dim-3 strain, as demonstrated by Southern blotting (Fig. 2A) and genome-wide bisulfite sequencing (BS-Seq; Figs. 1B and S1), but has no marked effect on DNA methylation levels in a dim-3+ strain. We tested several possibilities to determine the source of this histidine effect. The "cross pathway control" system causes de-repression of several amino acid biosynthetic pathways concomitant with changes in the level of an individual amino acid [25]. We tested its involvement in the histidine effect by checking for possible consequences of added arginine, lysine, and tryptophan, but found they neither caused loss of DNA methylation nor restored it when added with histidine (S4 Fig.). RNAi components are not required for DNA methylation [11], but histidine supplementation and exposure to other DNA damaging agents, induces expression of the Neurospora Argonaute QDE-2 (EAA31129.2) and increases the levels of qiRNAs (QDE-2-interacting small RNAs; [26]). We tested the possible involvement of DNA damage and RNAi in the histidine effect by growing a dim-3 strain in medium containing histidine (his), hydroxyurea (HU), or ethyl methane sulfonate (EMS). Only histidine addition reduced DNA methylation in dim-3 strains (S5A Fig.), and this effect also occurred in a Δqde-2 strain (S5B Fig.), indicating the histidine effect in dim-3 strains involves neither DNA damage nor qiRNAs. Considering that NUP-6 is critical for nuclear transport and that histidine does not further deplete H3K9me3 levels (below), we investigated whether the histidine effect results from altered nuclear transport and/or sub-nuclear localization of DIM-2 (AF348971.1) or HP1 (AY363166.1), which operate downstream of H3K9me3. We found no significant difference in levels of DIM-2-3xFLAG in nuclei from a dim-3 strain grown in the presence or absence of histidine relative to WT (Fig. 2B), suggesting that neither histidine nor the nup-6dim-3 allele impacts DIM-2 nuclear shuttling. In addition, cytological analyses of HP1-GFP (S6 Fig.; [11]) and DamID analyses of HP1-DAM (below) revealed no evidence of a transport defect in dim3 strains. We next tested if histidine perturbs sub-nuclear targeting of DIM-2 or HP1 in a dim-3 strain. The localization of many heterochromatin-specific proteins, including DIM-2, are not readily detected by standard chromatin immunoprecipitation (ChIP), perhaps due to transient chromatin interactions, but can be detected by DamID [8]. Therefore, we fused the DNA adenine methyltransferase (dam) gene to the downstream ends of the dim-2 [27] and hpo (encoding HP1) genes, expressed these constructs in dim-3 and dim-3+ strains grown in the presence or absence of supplemented histidine, and tested their localization by digestion of genomic DNA with the GAmTC-specific restriction endonuclease DpnI followed by Southern blotting. DIM-2-DAM was found to localize to heterochromatin in both dim-3+ and dim-3 strains grown in minimum medium (Figs. 2C and S7A). In contrast, in a dim-3 strain grown with histidine, DIM-2-DAM showed reduced localization to heterochromatin, while histidine did not alter DIM-2-DAM localization in a dim-3+ strain (Figs. 2C and S7A). Unlike the situation with DIM-2, histidine did not reduce, and in fact slightly increased, the heterochromatic localization of HP1-DAM (Figs. 2D and S7B). These findings suggest that in dim-3 strains, histidine may compromise the direct interaction between DIM-2 and HP1 that is necessary for DNA methylation in Neurospora [10].

The dim-3 strain has a global reduction of H3K9me3 To determine whether the reduced DNA methylation observed in dim-3 strains reflects a loss of H3K9me3, we assessed global H3K9me3 levels by western blotting. H3K9me3 was greatly


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Fig 2. Histidine supplementation exacerbates loss of DNA methylation in dim-3 strains by displacing DIM-2 from heterochromatin. [A] Southern blot assay (as in Fig. 1A) of DNA methylation in dim-3+ (WT) or dim-3 prototrophic strains grown in minimal or histidine supplemented medium. [B] (left) α-FLAG western blots showing DIM-2-3xFLAG levels in WT and dim-3 nuclei grown in medium with or without histidine compared with a loading control (histone H3, [hH3]); (right) Quantification of DIM-2-3xFLAG levels from three independent experiments, normalized to hH3 levels. [C-D] Southern blot assay of DamID experiments with WT and dim-3 strains containing [C] DIM-2-DAM or [D] HP1-DAM, grown with or without histidine, and probed for the heterochromatic region 2:B3. Genomic DNA was digested with DpnI (DI), specifically cutting GAmTC sequences or with the Am-insensitive isoschizomer DpnII (DII) to monitor complete digestion. doi:10.1371/journal.pgen.1005083.g002


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Fig 3. Global reduction of H3K9me3 in dim-3compromises telomeric silencing. [A] Western blots of histones isolated from indicated strains were probed for H3K9me3, or histone H3 (hH3) as a loading control. [B] (Left) Western blots of histones isolated from the indicated strains grown with or without histidine probed for H3K9me3 or hH3. (Right) Quantification of H3K9me3 levels from three independent experiments, normalized to hH3 levels. [C] Chromatin Immunoprecipitation sequencing (ChIP-seq) of H3K9me3 from dim-3+ (WT) [56] and dim-3 strains displayed on IGV, as in Fig. 1B. Due to the vastly reduced trimethylation of H3K9 in the dim-3 strain, this strain shows a reduced signal-to-noise ratio, rendering background more prominent. [D] Growth of strains containing the telVR::hph or cenVIR::bar reporter cassettes on minimum medium (MIN) plates or plates supplemented with hygromycin (200 μg/mL, +HYG, left) or phosphinothricin (8 mg/mL, +BASTA, right). Approximate numbers of spotted conidia indicated below the pictures. doi:10.1371/journal.pgen.1005083.g003

reduced in dim-3 strains compared to that in a WT strain, and was reestablished after introduction of an ectopic nup-6+ gene (Fig. 3A), indicating that NUP-6 is required for normal levels of H3K9me3. Considering that added histidine reduced DNA methylation levels in dim-3 strains (Fig. 2), we checked if histidine exacerbates the H3K9me3 loss. Western blots showed no additional loss of H3K9me3 (Fig. 3B), supporting the notion that histidine reduces DNA methylation by compromising DIM-2 localization in a dim-3 background. To determine if the residual H3K9me3, like DNA methylation, is found in normal heterochromatic regions in dim-3 strains, we carried out H3K9me3-specific ChIP with high throughput sequencing of associated DNA (ChIP-seq), and despite low signal, found an apparently equivalent distribution of this chromatin mark as in a dim-3+ strain, indicating that the remaining H3K9me3 is correctly localized to heterochromatin in a dim-3 strain (Figs. 3C and S8). Presumably, decreased DNA methylation in dim-3 strains is due to the dramatic reduction of H3K9me3 in heterochromatin.

The dim-3 mutation causes loss of telomeric silencing To determine if the H3K9me3 loss in dim-3 strains might compromise heterochromatin-associated silencing, we tested the expression of drug resistance markers integrated at telomeric (telVR::hph) and centromeric (cenVIR::bar) sites, which are silent even in the absence of DNA methylation, i.e. in a dim-2 mutant [27,28]. The dim-3 allele de-repressed the telVR::hph


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marker as evidenced by growth in the presence of hygromycin (Fig. 3D), indicating that heterochromatin in dim-3 is compromised, although this de-repression is not as striking as in an hpo mutant, which fully de-represses the marker [28]. The dim-3 allele did not de-repress the cenVIR::bar marker (Fig. 3D), consistent with the existence of a DNA methylation-independent mechanism for silencing centromeric heterochromatin [27].

Nuclear transport of the H3K9me3 machinery is not deficient in dim-3 strains Considering that the canonical function of NUP-6 is to transport proteins into the nucleus, we tested if a dim-3 strain has diminished nuclear transport of proteins required for heterochromatin formation and of a protein known to depend on the Importin α/β import system. Our observations that DIM-2 and HP1 localize normally in the nucleus (Figs. 2B and S6) and that dim-3 strains have only a minor growth defect (S2C Fig.) did not support the idea that dim-3 strains are defective in nuclear shuttling. Nevertheless, because the dim-3 mutant shows a striking loss of H3K9me3, we tested whether nuclear transport of DCDC components (DIM-5 [AF419248.1], DIM-7 [AL513463.1], DDB1 [EAA33111.1], DIM-9 [XP_956278.2], and CUL4 [XP_957743.2]) is impaired. To monitor nuclear transport, nuclei were isolated from dim-3+ and dim-3 strains bearing DCDC members individually 3xFLAG-tagged at their endogenous loci [8,15] and protein levels were assessed by western blotting (Figs. 4A and S9). Nuclei preparations were shown to be clean of cytoplasmic contamination by probing western blots for phosphoglycerate kinase (α-PGK, EAA33194.1; Fig. 4B). We found that the nuclear level of every component of DCDC was undiminished by the dim-3 mutation (Figs. 4A and S9). Indeed, some of the components actually showed increased nuclear abundance. DIM-5 and DIM-7 showed equivalent levels in dim-3+ and dim-3 strains while the nuclear levels of DDB1, DIM-9 and CUL4 were higher in the dim-3 strain (Figs. 4A and S9). Moreover, an examination of the distribution of DCDC components between the nuclear and cytoplasmic fractions revealed no increase in the relative amount of the proteins in the cytoplasmic fraction (Fig. 4B). DIM-7, which is an exclusively nuclear protein, showed no change in abundance or localization. Similarly, while the nuclear level of DIM-5 increased slightly in the dim-3 strain (Fig. 4A), the relative nuclear/cytoplasmic distribution of the protein was equivalent in dim-3+ and dim-3 strains. DIM-9, which is predominantly nuclear in dim-3+ strains, showed an increase in both the cytoplasmic and nuclear fractions of the dim-3 strain (Fig. 4B). It is interesting that the nuclear levels of DDB1, DIM-9, and CUL4 were elevated in dim-3 strains. The increase in CUL4 only occurred when this strain is supplemented with histidine (compare Figs. 4B and S9), which is consistent with the induction of a DNA damage response, as previously documented [26]. The basis for increased DDB1 and DIM-9 levels in dim-3 strains is not clear but may reflect a feedback mechanism to regulate the amount of functional DCDC. Results of qRT-PCR analyses of dim-7, ddb1, and dim-9 transcripts in dim-3+ and dim-3 strains did not reveal RNA differences that could account for the differences in protein levels (S10 Fig.), suggesting the effect is at the translational or posttranslational level. To test if the dim-3 mutation impacted nuclear transport of a protein with a nuclear localization signal (NLS) known to be bound and transported by Importin α, we overexpressed a GFP reporter construct with an N-terminal SV40 monopartite NLS [22,29–32] in dim-3+ and dim-3 strains and examined nuclear GFP signal by fluorescence microscopy. We found that 91.6% of dim-3+ cells had strong GFP signal inside their nuclei, and this result was mirrored in a dim-3 strain, where 92.5% of cells had nuclear GFP (Fig. 4C). Thus, NUP-6dim-3 effectively transports canonical nuclear cargo.


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Fig 4. Nuclear transport of DCDC components is unaffected by the dim-3 mutation. [A] α-FLAG and αhistone H3 (hH3; loading control) western blots of dim-3+ (WT) or dim-3 nuclei expressing individually FLAGtagged DCDC components. All strains were grown in the presence of histidine. Average and standard deviation of nuclear FLAG-tagged protein levels of three experiments are indicated below the representative images shown. [B] α-FLAG western blots of the total (T) proteins, cytoplasmic (C) fraction, and nuclear (N) fraction of nuclei preparations from dim-3+ (WT) and dim-3 strains expressing DIM-5-3xFLAG, DIM-73xFLAG, or DIM-9-3xFLAG. Representative α-histone H3 (hH3) and α-PGK blots from the DIM-5-3xFLAG nuclei are shown as nuclear and cytoplasmic fraction controls, respectively; equivalent results were obtained for all nuclei preparations. [C] Representative differential interference contrast (DIC) and GFP fluorescent images of (left) dim-3+ or (right) dim-3 strains expressing an overexpressed NLSSV40-GFP reporter construct (pCCG::NLSSV40::LexADBD::GFP). While just one representative, multinucleate cell is displayed in the figures here and elsewhere in the paper, we visualized numerous vegetative cells, including both conidia and hyphal cells [62]. Percentages of conidia exhibiting each pattern are listed on the right (P value = 0.43, X2test). Scale bar indicates 5μm. doi:10.1371/journal.pgen.1005083.g004

Interactions within the DCDC are not decreased in dim-3 strains Given that all DCDC members, as well as DIM-2 and HP1, are transported into the nucleus in dim-3 strains, we next considered the possibility that NUP-6dim-3 somehow interferes with DCDC assembly. To test this hypothesis, we monitored DCDC component interactions in


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Fig 5. Interactions between DCDC members are not compromised by the mutations in dim-3. [A] Western blots detecting DIM-5 interaction with DIM-73X-FLAG or DIM-9-3XFLAG from dim-3+ (WT) or dim-3 nuclei. For quantification of purified DIM-5, levels of DIM-7/9-3xFLAG IP from WT and dim-3 nuclei were equalized and the level of purified DIM-5 from dim-3 was normalized to the adjusted DIM-7/9-3xFLAG IP level from dim-3. The experiment performed in triplicate, and average percent and standard deviation of wild type DIM-5 is noted below. [B] Western blots detecting DIM-9-3xHA interaction with purified DIM-8-3X-FLAG from dim-3+ (WT) or dim-3 nuclei. Experiment performed in duplicate and normalized as in [A], with average noted. Control experiment with DIM-9-3xHA co-IP from WT and dim-3 nuclei without FLAG-tagged protein demonstrates α-FLAG IP specificity. The star indicates a non-specific band routinely detected in experiments with Neurospora nuclear extracts probed with the rabbit-derived FLAG antibody. [C] Western blots detecting DIM-9-3xHA interaction with purified DIM-7-3X-FLAG from dim-3+ (WT) or dim-3 nuclei. The experiment was performed in triplicate and normalized as in [A]. doi:10.1371/journal.pgen.1005083.g005

dim-3 and dim-3+ nuclei by co-immunoprecipitation (co-IP) assays with 3xFLAG-tagged DCDC members. We began by analyzing the interaction between DIM-7 and DIM-5. Equivalent levels of DIM-7-3xFLAG were recovered from dim-3 and dim-3+ nuclei (Fig. 5A), consistent with our finding that DIM-7 levels in the nucleus are not affected by the dim-3 mutation (Figs. 4A and S9). Equivalent levels of DIM-5 were found in co-IPs of both backgrounds, implying that DIM-7 binding to DIM-5 is not compromised in dim-3 cells (Fig. 5A). Since the DIM-5 interaction with the DCDC members appears mediated through DIM-7 [15], we assessed DIM-5 binding to DIM-9-3xFLAG in dim-3+ and dim-3 nuclei. Because more DIM-93xFLAG was recovered from dim-3 nuclei than from dim-3+ nuclei (Fig. 5A), we normalized


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the amount of DIM-5 purified to the DIM-9-3xFLAG bait levels. The DIM-5 level directly correlated with the DIM-9-3xFLAG level (Fig. 5A), indicating that DIM-5 is not limiting and this interaction is not compromised in dim-3 cells. We also monitored DIM-9-3XHA binding to DDB1-3xFLAG. Equal levels of DDB13xFLAG were purified from dim-3+ and dim-3 nuclei, and the DIM-9-3xHA interaction appeared normal (Fig. 5B), suggesting no disruption in DCAF-substrate adaptor binding by the dim-3 mutation. We then monitored the nuclear interaction between DIM-7-3xFLAG and DIM-9-3xHA, which were previously shown to directly interact [15]. DIM-9-3xHA showed an increased interaction with DIM-7-3xFLAG in dim-3 nuclei (Fig. 5C), potentially because DIM9 levels are increased in dim-3 strains (Fig. 5C, input). In summary, all examined DCDC members showed normal or increased interactions in dim-3 nuclei, implying that the H3K9me3 loss is not due to impaired DCDC assembly in dim-3 strains.

DIM-5 and DIM-7 are mislocalized from heterochromatin in dim-3 strains It remained possible that the DCDC is not properly localized to heterochromatin in dim-3 strains. To investigate this possibility, we examined the localization of DIM-5, DIM-7, and DIM-9 in dim-3 strains by DamID. DIM-5-DAM was previously shown to localize to heterochromatin, and this localization was dependent on DIM-7 [8]. We found that the dim-3 mutation caused substantially reduced localization of DIM-5-DAM at the three representative heterochromatic regions tested (8:A6, 2:B3, and 8:G3; Figs. 6A and S11A). This reduction was not exacerbated by histidine (S11B Fig.), in contrast to the case for DIM-2 localization (Fig. 2). Probing for euchromatin regions (hH3 and pan-1) revealed that DIM-5-DAM is not misdirected to euchromatin (Figs. 6A and S11A). The C-terminus of DIM-7 is critical for normal function [8], such that DAM-tagged DIM-7 does not fully complement dim-7 mutations. Nevertheless, as with DIM-5-DAM, we found that association of DIM-7-DAM with the heterochromatin regions (8:A6, 2:B3, and 8:G3) was also markedly reduced in the dim-3 background (Figs. 6B and S11C), suggesting that NUP6dim-3 impacts targeting of DIM-7-DAM to heterochromatin. In contrast, DamID of DIM-9DAM did not reveal marked differences between the dim-3+ and dim-3 strains (Figs. 6B and S11C). We note that DIM-7-DAM and DIM-9-DAM may show a greater association with the euchromatic gene tested (hH3) than did DIM-5-DAM, perhaps reflecting an unknown role of these proteins outside of heterochromatin. The interaction of DIM-7-DAM, but not DIM-9DAM, with the euchromatin marker was reduced in the dim-3 strain (Figs. 6A and S11C). To confirm that DIM-7-DAM is mislocalized, we expressed DIM-7-GFP in dim-3+ and dim-3 vegetative tissue. The level of DIM-7-GFP produced from the native dim-7 promoter was not cytologically detectable, leading us to express it under the control of the stronger ccg-1 promoter. Despite overexpression, DIM-7-GFP co-localized with HP1-mCherry (S12 Fig.), at least in the cells examined (conidia), suggesting that DIM-7-GFP behaves normally to mark heterochromatic regions in vivo despite the importance of the DIM-7 C-terminus [8]. In 93.5% of dim-3+ cells DIM-7-GFP formed compact foci, typically at or near the nuclear periphery (Figs. 6C and S12), consistent with DIM-7-GFP marking heterochromatic regions. Interestingly, approximately half of dim-3 nuclei examined also showed such foci (Fig. 6D). It would be interesting to learn whether the observed difference between dim-3 and dim-3+ cells reflect differences in cell cycles of these strains. Unfortunately, because no genetic method to synchronize Neurospora has been developed, our studies were limited to unsynchronized cells. DIM-7GFP appears equivalently expressed and transported into nuclei of dim-3+ and dim-3 strains (S13 Fig.). Thus, the dim-3 mutation seems to partially perturb normal localization of DIM-7 fusion proteins within the nucleus.


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Fig 6. DIM-5 and DIM-7 are mislocalized from heterochromatin in a dim-3 strain. [A] DamID experiments, as in Fig. 3, with two independent samples of dim-3+ (WT) and dim-3 strains expressing DIM-5-DAM. Representative blots from two heterochromatic regions (8:A6 and 2:B3) and one euchromatic region (hH3, encoding histone H3, EAA26767.1) are shown. [B] DamID experiments with DIM-7-DAM and DIM-9-DAM. [C] Representative differential interference contrast (DIC), GFP fluorescent, Hoechst 33342-stained DNA, and merged images of dim+ strains bearing Pccg::DIM-7-GFP. Each panel displays one conidium, with four nuclei (top) or two nuclei (bottom) visualized. [D] Representative DIC, GFP, Hoechst 33342-stained DNA, and merged images of dim-3 strains with Pccg::DIM-7-GFP; each panel displays one conidium showing two nuclei. Percentages of cells containing at least one sub-nuclear focus are listed on right, and the P-value for loss of focus formation is